3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labeled proteins

Summary The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses ¹³C–¹⁵N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the ¹³C and ¹⁵N nuclei, thereby providing the four resonance frequencies of the -proton, the -carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in ¹³C-¹⁵N-double-labeled proteins.Abbreviations 3D, 4D three-dimensional, four-dimensional - TPPI time-proportional phase incrementation - ct constant-time - rf radiofrequency - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - glutaredoxin(C14S) mutant E. coli glutaredoxin with the cysteine at position 14 replaced by serine